1. Field of the Invention
The present invention is in the fields of cell biology, immunology and oncology. Specifically, the invention relates to humanized antibodies that recognizes αvβ6 integrins which comprises a variable region of nonhuman origin and at least—a portion of an immunoglobulin of human origin. The invention also relates to processes for their preparation, to pharmaceutical compositions comprising them and to methods of treating various diseases by administering humanized anti-αvβ6 antibodies. The invention also relates to the identification of differential expression of the integrin αvβ6 on the surfaces of tumor cells and tissues, the use of this differential expression in determining the metastatic potential of tumor cells, and methods of diagnosis and treatment/prevention of tumor metastasis and for elimination of residual metastatic tumor cells using ligands, particularly antibodies, that bind to integrin αvβ6.
2. Related Art
Integrins are cell surface glycoprotein receptors which bind extracellular matrix proteins and mediate cell-cell and cell-extracellular matrix interactions (generally referred to as cell adhesion events) (Ruoslahti, E., J. Clin. Invest. 87:1-5 (1991); Hynes, R. O., Cell 69:11-25 (1992)). These receptors are composed of noncovalently associated alpha (α) and beta (β) chains which combine to give a variety of heterodimeric proteins with distinct cellular and adhesive specificities (Albeda, S. M., Lab. Invest. 68:4-14 (1993)). Recent studies have implicated certain integrins in the regulation of a variety of cellular processes including cellular adhesion, migration, invasion, differentiation, proliferation, apoptosis and gene expression (Albeda, S. M., Lab. Invest. 68:4-14 (1993); Juliano, R., Cancer Met. Rev. 13:25-30 (1994); Ruoslahti, E. and Reed, J. C., Cell 77:477-478 (1994); and Ruoslahti, E. and Giancotti, F. G., Cancer Cells 1:119-126 (1989); Plow, Haas et al. 2000; van der Flier and Sonnenberg 2001).
The αvβ6 receptor is one member of a family of integrins that are expressed as cell surface heterodimeric proteins (Busk, M. et al., J. Biol. Chem. 267(9):5790-5796 (1992)). While the αv subunit can form a heterodimer with a variety of β subunits (β1, β3, β5, β6 and β8), the β6 subunit can only be expressed as a heterodimer with the αv subunit. The αvβ6 integrin is known to be a fibronectin-, latency associated peptide (LAP)- and tenascin C-binding cell surface receptor, interacting with the extracellular matrix through the RGD tripeptide binding sites thereon (Busk, M. et al., J. Biol. Chem. 267:5790-5796 (1992); Weinacker, A. et al., J. Biol. Chem. 269:6940-6948 (1994); Prieto, A. L. et al., Proc. Natl. Acad. Sci. USA 90:10154-10158 (1993)). Although the αvβ6 integrin was first identified and sequenced more than 10 years ago, the biological significance of αvβ6, especially in disease, is still under investigation. The expression of αvβ6 is restricted to epithelial cells where it is expressed at relatively low levels in healthy tissue and significantly upregulated during development, injury, and wound healing (Breuss, J. M. et al., J. Histochem. Cytochem. 41:1521-1527 (1993); Breuss, J. M. et al., J Cell Sci. 108:2241-2251 (1995); Koivisto, L. et al., Cell Adhes. Communic. 7:245-257 (1999); Zambruno, G. et al., J. Cell Biol. 129(3):853-865 (1995); Hakkinen, L. et al., J. Histochem. Cytochem. 48(6):985-998 (2000)). An increasing number of recent reports demonstrate that αvβ6 is upregulated on cancers of epithelial origin, including colon carcinoma (Niu, J. et al, Int. J. Cancer 92:40-48 (2001); Bates, R. C. et al., J. Clin. Invest. 115:339-347 (2005)), ovarian cancer (Ahmed, N. et al., J. Cell. Biochem. 84:675-686 (2002); Ahmed, N. et al., J. Histochem. Cytochem. 50:1371-1379 (2002); Ahmed, N. et al., Carcinogen. 23:237-244 (2002)), squamous cell carcinoma (Koivisto, L. et al., Exp. Cell Res. 255:10-17 (2000); Xue, H. et al., Biochem. Biophys. Res. Comm. 288:610-618 (2001); Thomas, G. J. et al., J. Invest. Dermatol. 117:67-73 (2001); Thomas, G. J. et al., Int. J. Cancer 92:641-650 (2001); Ramos, D. M. et al., Matrix Biol. 21:297-307 (2002); (Agrez, M. et al., Br. J. Cancer 81:90-97 (1999); Hamidi, S. et al., Br. J. Cancer 82(8):1433-1440 (2000); Kawashima, A. et al., Pathol. Res. Pract. 99(2):57-64 (2003)), and breast cancer (Arihiro, K. et al., Breast Cancer 7:19-26 (2000)). It has also been reported that the αv subunit may be involved in tumor metastasis, and that blocking this subunit consequently may prevent metastasis (for review, see Imhof, B. A. et al., in: “Attempts to Understand Metastasis Formation I,” U. Günthert and W. Birchmeier, eds., Berlin: Springer-Verlag, pp. 195-203 (1996)).
The αvβ6 integrin may have multiple regulatory functions in tumor cell biology. Recent studies have demonstrated that the extracellular and cytoplasmic domains of the β6 subunit mediate different cellular activities. The extracellular and transmembrane domains have been shown to mediate TGF-β activation and adhesion (Sheppard, D., Cancer and Metastasis Rev. 24:395-402 (2005); Munger, J. S. et al., Cell 96:319-328 (1999)). The cytoplasmic domain of the β6 subunit contains a unique 11-amino acid sequence that is important in mediating αvβ6 regulated cell proliferation, MMP production, migration, and pro-survival (Li, X. et al., J. Biol. Chem. 278(43):41646-41653 (2003); Thomas, G. J. et al., J. Invest. Derm. 117(1):67-73 (2001); Thomas, G. J. et al., Br. J. Cancer 87(8):859-867 (2002); Janes, S. M. and Watt, F. M., J. Cell Biol 166(3):419-431 (2004)). The β6 subunit has been cloned, expressed and purified (Sheppard et al., U.S. Pat. No. 6,787,322 B2, the disclosure of which is incorporated herein by reference in its entirety), and function-blocking antibodies that selectively bind to the αvβ6 integrin have been reported (Weinreb et al., J. Biol. Chem. 279:17875-17877 (2004), the disclosure of which is incorporated herein by reference in its entirety). Antagonists of αvβ6 (including certain monoclonal antibodies) have also been suggested as possible treatments for certain forms of acute lung injury and fibrosis (see U.S. Pat. No. 6,692,741 B2 and WO 99/07405, the disclosures of which are incorporated herein by reference in their entireties).
αvβ6 can bind to several ligands including fibronectin, tenascin, and the latency associated peptide-1 and -3 (LAP1 and LAP3), the N-terminal 278 amino acids of the latent precursor form of TGF-β1 through a direct interaction with an arginine-glycine-aspartate (“RGD”) motif (Busk, M. et al., J. Biol. Chem. 267(9):5790-5796 (1992); Yokosaki, Y. et al., J. Biol. Chem. 271(39):24144-24150 (1996); Huang, X. Z. et al., J. Cell. Sci. 111:2189-2195 (1998); Munger, J. S. et al., Cell 96:319-328 (1999)). The TGF-β cytokine is synthesized as a latent complex which has the N-terminal LAP non-covalently associated with the mature active C-terminal TGF-β cytokine. The latent TGF-β complex cannot bind to its cognate receptor and thus is not biologically active until converted to an active form (Barcellos-Hoff, M. H., J. Mamm. Gland Biol. 1(4):353-363 (1996); Gleizes, P. E. et al., Stem Cells 15(3):190-197 (1997); Munger, J. S. et al., Kid. Int. 51:1376-1382 (1997); Khalil, N., Microbes Infect. 1(15):1255-1263 (1999)). αvβ6 binding to LAP1 or LAP3 leads to activation of the latent precursor form of TGF-β1 and TGF-β3 (Munger, J. S. et al., Cell 96:319-328 (1999)), proposed as a result of a conformational change in the latent complex allowing TGF-β to bind to its receptor. Thus, upregulated expression of αvβ6 can lead to local activation of TGF-β which in turn can activate a cascade of events downstream events.
The TGF-β1 cytokine is a pleiotropic growth factor that regulates cell proliferation, differentiation, and immune responses (Wahl, S. M., J. Exp. Med. 180:1587-1590 (1994); Massague, J., Annu. Rev. Biochem. 67:753-791 (1998); Chen, W. and Wahl, S. M., TGF-β: Receptors, Signaling Pathways and Autoimmunity, Basel: Karger, pp. 62-91 (2002); Thomas, D. A. and Massague, J., Cancer Cell 8:369-380 (2005)). The role that TGF-β1 plays in cancer is two-sided. TGF-β is recognized to tumor suppressor and growth inhibitory activity yet, many tumors evolve a resistance to growth suppressive activities of TGF-β1 (Yingling, J. M. et al., Nature Rev. Drug Discov. 3(12):1011-1022 (2004); Akhurst, R. J. et al., Trends Cell Biol. 11(11):S44-S51 (2001); Balmain, A. and Akhurst, R. J., Nature 428(6980):271-272 (2004)). In established tumors, TGF-β1 expression and activity has been implicated in promoting tumor survival, progression, and metastases (Akhurst, R. J. et al., Trends Cell Biol. 11(11):S44-S51 (2001); Muraoka, R. S. et al., J. Clin. Invest. 109(12):1551 (2002); Yang, Y. A. et al., J. Clin. Invest. 109(12):1607-1615 (2002)). This is postulated to be mediated by both autocrine and paracrine effects in the local tumor-stromal environment including the effects of TGF-β on immune surveillance, angiogenesis, and increased tumor interstitial pressure. Several studies have now shown the anti-tumor and anti-metastatic effects of inhibiting TGF-β1 (Akhurst, R. J., J. Clin. Invest. 109(12):1533-1536 (2002); Muraoka, R. S. et al., J. Clin. Invest. 109(12):1551 (2002); Yingling, J. M. et al., Nat. Rev. Drug Discov. 3(12):1011-1022 (2004); Yang, Y. A. et al., J. Clin. Invest. 109(12):1607-1615 (2002); Halder, S. K. et al., Neoplasia 7(5):509-521 (2005); Iyer, S. et al., Cancer Biol. Ther. 4(3):261-266 (2005)).
Increased expression of αvβ6 on tumors, particularly at the tumor-stromal interface, may reflect a unique mechanism for local activation of TGF-β1 and the ability to promote tumor survival, invasion, and metastasis. The high level of expression in human metastases infers a potential role for αvβ6 in establishing metastases which is consistent with previous reports that αvβ6 can mediate epithelial to mesenchymal transition, tumor cell invasion in vitro, and expression correlated with metastases in a mouse model (Bates, R. C. et al., J. Clin. Invest. 115(2):339-347 (2005); Thomas, G. J. et al., Br. J. Cancer 87(8):859-867 (2002); Morgan, M. R. et al., J. Biol. Chem. 279(25):26533-26539 (2004)).
We have previously described the generation of potent and selective anti-αvβ6 monoclonal antibodies (mAbs) that bind to both the human and murine forms of αvβ6 and block the binding of αvβ6 to its ligands and αvβ6 mediated activation of TGF-β1 (Weinreb, P. H. et al., J. Biol. Chem. 279(17):17875-17887 (2004)). As also described in PCT Publication WO 03/100033, herein incorporated in its entirety by reference, high affinity antibodies against αvβ6, including the identification and analysis of key amino acid residues in the complementary determining regions (CDRs) of such antibodies, were discovered and characterized. In particular, these high affinity antibodies (a) specifically bind to αvβ6; (b) inhibit the binding of αvβ6 to its ligand such as LAP, fibronectin, vitronectin, and tenascin with an IC50 value lower than that of 10D5 (International Patent Application Publication WO 99/07405); (c) block activation of TGF-β; (d) contain certain amino acid sequences in the CDRs that provide binding specificity to αvβ6; (e) specifically bind to the β6 subunit; and/or (f) recognize αvβ6 in immunostaining procedures, such as immunostaining of paraffin-embedded tissues.
WO 03/100033 also describes the discovery that antibodies that bind to αvβ6 can be grouped into biophysically distinct classes and subclasses. One class of antibodies exhibits the ability to block binding of a ligand (e.g., LAP) to αvβ6 (blockers). This class of antibodies can be further divided into subclasses of cation-dependent blockers and cation-independent blockers. Some of the cation-dependent blockers contain an arginine-glycine-aspartate (RGD) peptide sequence, whereas the cation-independent blockers do not contain an RGD sequence. Another class of antibodies exhibits the ability to bind to αvβ6 and yet does not block binding of αvβ6 to a ligand (nonblockers).
Furthermore, WO 03/100033 discloses antibodies comprising heavy chains and light chains whose complementarity determining regions (CDR) 1, 2 and 3 consist of certain amino acid sequences that provide binding specificity to αvβ6. WO 03/100033 also provides for antibodies that specifically bind to αvβ6 but do not inhibit the binding of αvβ6 to latency associated peptide (LAP) as well as antibodies that bind to the same epitope.
WO 03/100033 further discloses cells of hybridomas 6.1A8, 6.2B10, 6.3G9, 6.8G6, 6.2B1, 6.2A1, 6.2E5, 7.1G10, 7.7G5, and 7.1C5, isolated nucleic acids comprising a coding sequences and isolated polypeptides comprising amino acid sequences of the anti-αvβ6 antibodies. In particular, WO 03/100033 discloses anti-αvβ6 antibodies comprising heavy and light chain polypeptide sequences as antibodies produced by hybridomas 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10, 6.2A1, 6.2E5, 7.1G10, 7.7G5, or 7.1C5. Several of the hybridomas were deposited at the American Type Culture Collection (“ATCC”; P.O. Box 1549, Manassas, Va. 20108, USA) under the Budapest Treaty. In particular, hybridoma clones 6.3G9 and 6.8G6 were deposited on Aug. 16, 2001, and have accession numbers ATCC PTA-3649 and PTA-3645, respectively. The murine antibodies produced by hybridomas 6.3G9 and 6.8G6 are being further explored in the present application for their potential development as humanized antibodies.
The murine monoclonal antibody 3G9 is a murine IgG1, kappa antibody isolated from the β6 integrin −/− mouse (Huang et al., J. Cell Biol. 133:921-928 (1996)) immunized with human soluble αvβ6. The 3G9 antibody specifically recognizes the αvβ6 integrin epitope which is expressed at upregulated levels during injury, fibrosis and cancer (see, e.g., Thomas et al., J. Invest. Dermatology 117:67-73 (2001); Brunton et al., Neoplasia 3: 215-226 (2001); Agrez et al., Int. J. Cancer 81:90-97 (1999); Breuss, J. Cell Science 108:2241-2251 (1995)). It does not bind to other αv integrins and is cross-reactive to both human and murine molecules. The murine monoclonal antibody 3G9 has been described to block the binding of αvβ6 to LAP as determined by blocking of ligand binding either to purified human soluble αvβ6 or to β6-expressing cells, thereby inhibiting the pro-fibrotic activity of TGF-β receptor activation (see WO 03/100033). It has also been shown to inhibit αvβ6-mediated activation of TGF-β with an IC50 value lower than one of the known αvβ6 antibodies, 10D5 (Huang et al., J. Cell Sci. 111:2189-2195 (1998)).
The murine monoclonal antibody 8G6 is a murine IgG1, kappa antibody which also recognizes the αvβ6 integrin epitope, as described in WO 03/100033. The murine monoclonal antibody 8G6 is a cation-dependent, high affinity blocker of αvβ6 displaying the ability to inhibit αvβ6-mediated activation of TGF-β with an IC50 value lower than 10D5 (see WO 03/100033).
Both the 3G9 and 8G6 murine antibodies were effective in preventing fibrosis of the kidney and lung, as described in WO 03/100033. Furthermore, the murine antibody 3G9 was able to effectively inhibit tumor growth in a human tumor xenograft model, suggesting the potential role of αvβ6 in cancer pathology and the effectiveness of such blockade using antibodies directed at αvβ6.
Accordingly, there is a need to develop αvβ6 antibodies that are less antigenic in humans and that may be useful in the treatment of diseases involved in the αvβ6 pathway. With the advent of recombinant DNA methodology, it has become possible to structurally engineer antibody genes and produce modified antibody molecules with properties not obtainable by hybridoma technology. In the therapeutic arena, one aim of this methodology has been to reduce the immunogenicity in humans of rodent monoclonal antibodies by modifying their primary amino acid structure. Reduction of the immunogenicity of therapeutic antibodies is desirable because induction of an immune response can cause a spectrum of adverse effects in a patient, ranging from accelerated elimination of the therapeutic antibody, with consequent loss of efficacy, to fatal anaphylaxis at the most extreme.
One strategy to reduce immunogenicity of foreign monoclonal antibodies has been to replace the light and heavy chain constant domains of the monoclonal antibody with analogous domains of human origin, leaving the variable region domains of the foreign antibody intact. The variable region domains of the light and heavy chains are responsible for the interaction between the antibody and the antigen. Chimeric antibody molecules having mouse variable domains joined to human constant domains usually bind antigen with the same affinity constant as the mouse antibody from which the chimeric was derived. Such chimeric antibodies are less immunogenic in humans than their fully murine counterparts. Nevertheless, antibodies that preserve entire murine variable domains tend to provoke immune responses in a substantial fraction of patients.
That humans would mount an immune response to whole murine variable domains was predictable, thus, efforts to obtain variable domains with more human character had begun even before clinical trials of such standard chimeric antibodies had been reported. One category of methods frequently referred to as “humanizing,” aims to convert the variable domains of murine monoclonal antibodies to a more human form by recombinantly constructing an antibody variable domain having both mouse and human character. Humanizing strategies are based on several consensual understandings of antibody structure data. First, variable domains contain contiguous tracts of peptide sequence that are conserved within a species, but which differ between evolutionarily remote species, such as mice and humans. Second, other contiguous tracts are not conserved within a species, but even differ even between antibody producing cells within the same individual. Third, contacts between antibody and antigen occur principally through the non-conserved regions of the variable domain. Fourth, the molecular architecture of antibody variable domains is sufficiently similar across species that correspondent amino acid residue positions between species may be identified based on position alone, without experimental data.
Humanized strategies share the premise that replacement of amino acid residues that are characteristic of murine sequences with residues found in the correspondent positions of human antibodies will reduce the immunogenicity in humans of the resulting antibody. However, replacement of sequences between species usually results in reduction of antibody binding to its antigen. The art of humanization therefore lies in balancing replacement of the original murine sequence to reduce immunogenicity with the need for the humanized molecule to retain sufficient antigen binding to be therapeutically useful. This balance has been struck using two approaches.
In one approach, exemplified by U.S. Pat. No. 5,869,619, characteristically human residues are substituted for murine variable domain residues that are determined or predicted (i) to play no significant chemical role in the interaction with antigens and (ii) to be positioned with side chains projecting into the solvent. Thus, exterior residues remote from the antigen binding site are humanized, while interior residues, antigen binding residues, and residues forming the interface between variable domains remain murine. One disadvantage of this approach is that rather extensive experimental data is required to determine whether a residue plays no significant chemical role in antigen binding or will be positioned in the solvent in a particular three dimensional antibody structure.
In another more general approach, exemplified by U.S. Pat. No. 5,225,539, contiguous tracts of murine variable domain peptide sequence considered conserved are replaced with the correspondent tracts from a human antibody. In this more general approach, all variable domain residues are humanized except for the non-conserved regions implicated in antigen binding. To determine appropriate contiguous tracks for replacement, U.S. Pat. No. 5,225,539 utilized a classification of antibody variable domain sequences that had been developed previously by Wu and Kabat, J Exp Med. 132(2):211-250 (1970).
Wu and Kabat pioneered the alignment of antibody peptide sequences, and their contributions in this regard were several-fold: First, through study of sequence similarities between variable domains, they identified correspondent residues that to a greater or lesser extent were homologous across all antibodies in all vertebrate species, inasmuch as they adopted similar three-dimensional structure, played similar functional roles, interacted similarly with neighboring residues, and existed in similar chemical environments. Second, they devised a peptide sequence numbering system in which homologous immunoglobulin residues were assigned the same position number. One skilled in the art can unambiguously assign what is now commonly called Kabat numbering, to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. Third, for each Kabat-numbered sequence position, Kabat and Wu calculated variability, by which is meant the finding of few or many possible amino acids when variable domain sequences are aligned. They identified three contiguous regions of high variability embedded within four less variable contiguous regions. Other workers had previously noted variability approximately in these regions (hypervariable regions) and posited that the highly variable regions represented amino acid residues used for antigen binding. Kabat and Wu formally demarcated residues constituting these variable tracts, and designated these “complementarity determining regions” (CDRs), referring to chemical complementarity between antibody and antigen. A role in three-dimensional folding of the variable domain, but not in antigen recognition, was ascribed to the remaining less-variable regions, which are now termed “framework regions”. Fourth, Kabat and Wu established a public database of antibody peptide and nucleic acid sequences, which continues to be maintained and is well known to those skilled in the art.
The humanization method disclosed by U.S. Pat. No. 5,225,539 in using the Kabat classification results in a chimeric antibody comprising CDRs from one antibody and framework regions from another antibody that differs in species origin, specificity, subclass, or other characteristics. However, no particular sequences or properties were ascribed to the framework regions, indeed, U.S. Pat. No. 5,225,539 taught that any set of frameworks could be combined with any set of CDRs. Framework sequences have since been recognized as being important for conferring the three dimensional structure of an antibody variable region necessary to retain good antigen binding. Subsequent developments in the field have been refinements within the scope of U.S. Pat. No. 5,225,539 to deal with loss of avidity for antigen observed with some humanized antibodies relative to the avidity of the corresponding mouse antibodies.
U.S. Pat. No. 5,693,761 discloses one refinement on U.S. Pat. No. 5,225,539 for humanizing antibodies, and is based on the premise that ascribes avidity loss to problems in the structural motifs in the humanized framework which, because of steric or other chemical incompatibility, interfere with the folding of the CDRs into the binding-capable conformation found in the mouse antibody. To address this problem, U.S. Pat. No. 5,693,761 teaches using human framework sequences closely homologous in linear peptide sequence to framework sequences of the mouse antibody to be humanized. Accordingly, the methods of U.S. Pat. No. 5,693,761 focus on comparing framework sequences between species. Typically, all available human variable domain sequences are compared to a particular mouse sequence and the percentage identity between correspondent framework residues is calculated. The human variable domain with the highest percentage is selected to provide the framework sequences for the humanizing project. U.S. Pat. No. 5,693,761 also teaches that it is important to retain in the humanized framework, certain amino acid residues from the mouse framework critical for supporting the CDRs in a binding-capable conformation.
In other approaches, criticality of particular framework amino acid residues is determined experimentally once a low-avidity humanized construct is obtained, by reversion of single residues to the mouse sequence and assaying antigen binding as described by Riechmann et al., Nature 332(6162):323-327 (1988). Another example approach for identifying criticality of amino acids in framework sequences is disclosed by U.S. Pat. No. 5,821,337 and U.S. Pat. No. 5,859,205. These references disclose specific Kabat residue positions in the framework, which, in a humanized antibody may require substitution with the correspondent mouse amino acid to preserve avidity. Accordingly, the resulting frameworks constructed, which are part human and part mouse, still frequently exhibit human immunogenicity or lowered antigen binding, thereby requiring numerous iterations in framework construction to obtain a suitable framework for therapeutic uses.
There is therefore, a need in the art to develop αvβ6 antibodies that are less antigenic in humans. The present invention provides for the generation of humanized antibodies that are specifically reactive with αvβ6. The present invention also provides methods for making such humanized antibodies by providing humanized antibodies that reliably identify suitable human framework sequences to support non-human CDR regions and further to provide humanized antibodies that retain high antigen binding with low immunogenicity in humans. The present invention also provides for uses of such humanized antibodies reactive with αvβ6 in the treatment, diagnosis and/or prevention of various diseases and disorders.